Analysis of the Long-Term Impact on Cellular Immunity in COVID-19-Recovered Individuals Reveals a Profound NKT Cell Impairment

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affected over 120 million people and killed over 2.7 million individuals by March 2021. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remain to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19-convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2-unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of annexin V and 7-aminoactinomycin D (7-AAD) double-positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies and TIM-3 expression on CD4 and CD8 T cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by granzyme B (GzmB) expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully able to proliferate and produce effector cytokines upon T cell receptor (TCR) stimulation. Collectively, we provide a comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease.IMPORTANCE Wuhan was the very first city hit by SARS-CoV-2. Accordingly, the patients who experienced the longest phase of convalescence following COVID-19 reside here. This enabled us to investigate the "immunological scar" left by SARS-CoV-2 on cellular immunity after recovery from the disease. In this study, we characterized the long-term impact of SARS-CoV-2 infection on the immune system and provide a comprehensive picture of cellular immunity of a convalescent COVID-19 patient cohort with the longest recovery time. We revealed that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease; in particular, a profound NKT cell impairment was found in the convalescent phase of COVID-19.

Keywords: COVID-19; NKT cell; SARS-CoV-2; cellular immunity.

FIG 1

Characterization of immune cell subsets in individuals recovering from COVID-19. (A) The fold changes of the percentages (median) of total T cells, CD4 and CD8 T cells, B cells, NK cells, NKT-like cells, dendritic cells, and monocytes in the blood of CI (n = 30) compared to those of UI (n = 21) are depicted by radar plots. (B) The percentages of total T cells, CD4 and CD8 T cells, B cells, NK cells, and monocytes in the blood of UI (n = 21) and CI (n = 30) were analyzed by flow cytometry. (C) The absolute numbers and percentages of NKT-like cells in the blood of UI (n = 21) and CI (n = 30) were analyzed by flow cytometry. (D) The absolute numbers and percentages of invariant NKT (iNKT) cells in the blood of UI (n = 6) and CI (n = 19) were analyzed by flow cytometry. (E) The absolute numbers and percentages of dendritic cells in the blood of UI (n = 21) and CI (n = 30) were analyzed by flow cytometry. CI, COVID-19-convalescent individuals; UI, SARS-CoV-2-unexposed individuals. Statistically significant differences are indicated by asterisks (*, <0.05; **, <0.01; nonparametric Mann-Whitney test).

FIG 2

Characterization of immune cell death in individuals recovering from COVID-19. (A) The percentages of early apoptosis (annexin V⁺ 7-AAD⁻) and late apoptosis/necroptosis (annexin V⁺ 7-AAD⁺) of NKT-like cells in the blood of CI (n = 30) compared to those of UI (n = 21) were analyzed by flow cytometry. (B) Correlation analysis between the frequencies and the late apoptosis/necroptosis of NKT-like cells was performed in CI. (C) The percentages of early apoptosis (annexin V⁺ 7-AAD⁻) and late apoptosis/necroptosis (annexin V⁺ 7-AAD⁺) of CD4 T, CD8 T, B, and NK cells in the blood of CI (n = 30) compared to those of UI (n = 21) were analyzed by flow cytometry. CI, COVID-19-convalescent individuals; UI, SARS-CoV-2-unexposed individuals. Statistically significant differences are indicated by asterisks (*, <0.05; **, <0.01; ns, not significant; nonparametric Mann-Whitney test).

FIG 3

Characterization of T cell phenotypes in the PBMCs of individuals recovering from COVID-19. (A) The percentages of Ki67⁺ CD4 and CD8 T cells in the blood of UI (n = 21) and CI (n = 30) were analyzed by flow cytometry. (B and C) The fold changes of the percentages (median) of CD38⁺ HLA-DR⁻, CD38⁻ HLA-DR⁺, CD38⁺ HLA-DR⁺, PD-1⁺ TOX⁻, PD-1⁻ TOX⁺, PD-1⁺ TOX⁺, and TIM-3⁺ CD4 and CD8 T cells in the blood of CI (n = 30) compared to those of UI (n = 21) are depicted by radar plots. (D) The percentages of TIM-3⁺ CD4 and CD8 T cells in the blood of UI (n = 21) and CI (n = 30) were analyzed by flow cytometry. (E) The percentages of Foxp3⁺ CD4 T cells in the blood of UI (n = 21) and CI (n = 30) were analyzed by flow cytometry. CI, COVID-19-convalescent individuals; UI, SARS-CoV-2-unexposed individuals. Statistically significant differences are indicated by asterisks (*, <0.05; **, <0.01; nonparametric Mann-Whitney test).

FIG 4

Characterization of cytotoxic and cytokine profiles of T, NK, and NKT-like cells in the PBMCs of individuals recovering from COVID-19. (A) The fold changes of the percentages (median) of granzyme B-, perforin-, IFN-γ-, IL-6-, and GM-CSF-producing NKT-like cells in the blood of CI compared to those of UI are depicted by radar plots. (B) The percentages and MFI (geometric mean) of granzyme B expression of NKT-like cells in the blood of UI (n = 16) and CI (n = 25) were analyzed by flow cytometry. (C) The fold changes of the percentages (median) of granzyme B-, perforin-, IFN-γ-, IL-6-, and GM-CSF-producing CD8 T cells in the blood of CI compared to those of UI are depicted by radar plots. (D) The percentages and MFI (geometric mean) of granzyme B expression of CD8 T cells in the blood of UI (n = 21) and CI (n = 30) were analyzed by flow cytometry. (E) The fold changes of the percentages (median) of granzyme B-, perforin-, IFN-γ-, IL-6-, and GM-CSF-producing CD4 T cells in the blood of CI compared to those of UI are depicted by radar plots. (F) The percentages and MFI (geometric mean) of granzyme B expression of CD4 T cells in the blood of UI (n = 21) and CI (n = 30) were analyzed by flow cytometry. CI, COVID-19-convalescent individuals; UI, SARS-CoV-2-unexposed individuals. Statistically significant differences are indicated by asterisks (*, <0.05; **, <0.01; nonparametric Mann-Whitney test).

FIG 5

Characterization of the effector function of CD4 and CD8 T cells in the PBMCs of individuals recovering from COVID-19. PBMCs of UI (n = 5) and CI (n = 5) were either stimulated with anti-CD3 and anti-CD28 antibodies (αCD3/CD28) or left unstimulated (UC) and cultured for 5 days. The percentages of Ki67 (A)-, IFN-γ (B)-, IL-2 (C)-, and TNF-α (D)-positive CD4 (left) and CD8 (right) T cells were analyzed by flow cytometry. CI, COVID-19-convalescent individuals; UI, SARS-CoV-2-unexposed individuals. Statistically significant differences are indicated by asterisks (*, <0.05; **, <0.01; nonparametric Mann-Whitney test).